This invention relates to the field of biological studies and the like, having particular reference to studies observed or recorded over a period of time under controlled conditions and while under magnification. There are many instances where samples of biological material require study over a period of time and while under magnification. For example, a semen sample may require study to determine both the sperm count in the liquid medium of the sample and the motility of the sperm being observed. This may be done by providing a sample on a microscope slide and observing it under magnification of, say, 100x through a reference grid incorporated in the microscope objective. The grid may be divided into 100 squares and the sperm count in each of a representative number of squares may be made by a human observer to approximate the total number of sperm within the grid. Typically, the number of sperm observed within one square may be in the order of 100-200. Obviously, not every sperm in each square of the grid may be counted by the observer and a judicious selection is made as to which and how many of the squares are selected for accurate counting. The approximation is, therefore, highly subjective in nature. The other important factor to determine is sperm motility. This is determined by the observer by noting and counting the number of sperm which swim or are otherwise moving in the liquid medium within the selected and observed squares. The total number of sperm having such motility is again approximated to determine the percentage of the total which may be regarded as having motility.
In making the above determinations, it is essential that the volume of the semen sample observed in the confines of the grid be known and that the depth of such volumetric sample be such that the depth of the field of view permits all of the sperm within the confines of the grid to be observed. Although standard techniques have been developed to assure these factors during preparation of the slide sample, control over the factors which govern the volume of the sample confined to the grid area being observed and over deterioration of the sample is not uniform. Since body temperature is maintained in the sample during the study, evaporation of the liquid medium of the sample rapidly causes deterioration and it is difficult at best to prevent evaporation affecting the sample. In regard to this particular example, control over the location of the interface between the liquid medium and ambient air is important for control of evaporation. In accord with this invention, this control is effected by utilizing a miniaturized capillary environment which is wettable by the liquid medium of the sample. This is not easy to achieve because whereas many materials such as glass, for example, are wettable by water, they may not be sufficiently wettable by the biological liquid medium to achieve the desired and necessary miniaturized capillary environment. Mere selection of materials is inadequate because the desired wettability may not be present in any material unless it is specially prepared prior to use. That is, glass, for example, often and usually will possess surface film contamination which seriously affects its wettability characteristics and cannot be used as-received. Another problem is that a particular miniaturized capillary environment may require contiguous surface portions, one of which is highly wettable and the other of which is extremely hydrophobic. Again, mere selection of materials is inadequate and one may find that a conventional treatment of the miniaturized contiguous surfaces to control their surface energies or wettability characteristics results in chaos. For example, if the surface energy of one of the contiguous surfaces is to be increased while the other is to be decreased, conventional techniques may well result in an increase in both or a decrease in both so that the desired and correct combination of surface energies cannot be obtained.
Another example of biological study which may be desired is the study of a cell or a group or colony of cells, again in some liquid medium. Here, the volumetric consideration may not be so important as in the above example, but it is still a consideration because miniaturized chambers to accept the biological material should be so sized that some degree of physical confinement of the cells is effected. Moreover, control over surface energy or surface energies is equally if not more important than in the above example, particularly as the study involved may well require the presence of a gas environment as well as liquid nutrients for the cell or cells, all within the miniaturized capillary environment.
In one aspect, the invention concerns the method of making a miniaturized assembly to facilitate magnification study of biological samples in a liquid medium, which comprises the steps of: forming components which are inadequate as to wettability, relative to the liquid medium, to define a capillary environment containing the sample for a time sufficient to prevent deterioration of the sample while it is being studied; altering the wettability of the components relative to the liquid medium so that they may define a capillary environment containing the sample for a time sufficient to prevent deterioration of the sample while it is being studied; and assembling the components to define the capillary environment.
The invention disclosed herein is also directed to a miniaturized assembly to facilitate study of microscopic size particulate material contained in a medium while under magnification in a field of view having a particular depth of field, the assembly comprising the combination of plate means for defining a chamber having a portion which is to be within the field of view and is wettable by the medium to cause introduction and stabilization of the medium and the particulate material therewithin, and means for controlling the depth dimension of said portion of the chamber accurate to within 100 nanometers and the width dimension accurate to within 2 micrometers so as to correspond to the microscopic size of the particles and assure their disposition in the field of view. In terms of the study of semen as described above, the chamber containing the semen sample being observed may have a width dimension of 1.0 mm +or -2 micrometers and a depth dimension of 10 micrometers +or -100 nanometers. The width and depth dimensions assure an accurate determination of the volume being observed and the depth dimension is critical to assurance that all sperm being observed lie withinthe depth of field of the microscope under the magnification of interest.
More specifically, the invention relates to a system for microscopic evaluation of biological material contained in a field of view of a microscope, the biological material comprising discrete entities of the same kind dispersed in a medium, comprising the combination of first and second plates disposed in registry with each other, and means interposed between the plates for defining at least one biological evaluation chamber wettable by the medium and having a known set of dimensions which allows the determination of the concentration of entities in the field of view.
The invention also involves the method of making a miniature chamber assembly to facilitate study of microscopic size particulate material contained in a medium while under magnification which comprises the steps of providing two glass plates and forming a thin film of photoresist material on a surface of at least one plate in which the film is of a thickness of 0.25-250 micrometers, exposing the thin film to a patterned image and removing film material from the glass plate to leave discrete portions of the film in accord with the pattern and to expose the glass, altering the patterned film to render it either unwettable by the medium by exposing it to a fluorine plasma, or wettable by the medium by exposing it to an oxygen plasma or by selectively applying a thin film of aluminum, and superimposing the second glass plate upon the patterned film to form a system of miniaturized chambers between the plates and bounded by the patterned film.